The PCR in Practice

In order to decrease the likelihood of non-specific primer binding occurring during the initial (i.e. pre-first cycle) stage of the PCR thermocycling program (when the temperature is low enough to allow partial binding of the primer), one of the reaction components (usually the DNA polymerase or magnesium salt) may be omitted from the reaction mix and added once the initial target DNA melting temperature of 90–95°C is reached (PCR amplification will not proceed in the absence of magnesium ions). Unfortunately however, the simple act of opening and closing a PCR reaction tube greatly increases the chance of contamination of that tube. One elegant alternative to this problem is to physically separate the reaction components into two separate compartments, e.g. via a thin layer of wax. Magnesium chloride solution may then be added to the upper surface of the wax bead. The wax bead then melts at a temperature that is too high for non-specific binding of the PCR primers to the DNA template to occur. Some suppliers provide wax beads already impregnated with magnesium salt. Other elegant alternatives devised to decrease the likelihood of non-specific primer binding during the initial stage of the PCR thermocycling program, include heat-activatable thermostable DNA polymerases, which require approximately 10 minutes of heat activation at around 90°C before becoming activate. Other methods utilise polymerases coated with antibodies, which inhibit the action of the polymerase until the temperature is high enough to denature and remove the antibodies. All of these approaches are collectively named “hot-start” procedures (Chapter 7).

When performing multiple PCRs in a single “batch” (i.e. at the same time), it is advisable to prepare a “master mix” of reaction ingredients (comprising a multiple number of individual reaction mixes prepared in a single tube), and then pipette out the individual volumes into individual reaction tubes, rather than pipetting each separate component into every individual reaction tube one after the other. This process significantly reduces the number of pipetting actions that need to be performed and as such greatly contributes to the reproducibility and reliability of the PCR test protocol. The volume of a particular reagent required in a master mix is dependent on the number of PCRs to be performed, and may be calculated by multiplying the volume of the ingredient required for a single reaction, by the number of tests to be performed, including duplicates and positive and negative control samples. Additionally, for every master mix reaction prepared, an extra aliquot of each ingredient should be added (in order to account for volume errors inherent in any repeat pipetting action). Preferably, once prepared, the master mix should be immediately dispensed or stored on ice for short periods of time. Mastermixes and/ or individual PCR reaction mix aliquots may be stored frozen for a prolonged period of time and immediately thawed prior to use. In this case, it is preferable to prepare master mixes without thermostable DNA polymerase and then add the required volume of enzyme after thawing (immediately prior to PCR thermocycling). The time period during which frozen mastermix aliquots may be stored without loss of PCR amplification sensitivity should ideally be determined for each particular mix to be prepared and be routinely quality controlled [Kofler and Klausegger, 1999]. From the authors’ own experience, PCR master mix aliquots without thermostable DNA polymerase may be stored for 2–3 months at –20°C without loss of PCR sensitivity.